Members of the polo subfamily appear to play critical roles for cellular proliferation in eukaryotic organisms. They appear to play pivotal roles during the late G2/M phases of the cell cycle. We have previously shown that, in both budding yeast and cultured mammalian cells, the polo-box domain of polo kinases is essential and evolutionarily conserved in targeting the catalytic activity of the polo kinases to specific subcellular structures. In an effort to identify the polo-box-binding proteins, we carried out yeast two-hybrid screen using the polo-box domain of Plk1 (PBD) as a bait. The corresponding localization-incompetent polo-box triple mutant, PBD/FAA, was used as control. From this screen, we have isolated a novel centrosomal protein (we named hCenexin) and a novel kinetochores protein (Clone #17). Initial characterization with the transfected hCenexin showed that it primarily localizes to the mother centrioles. Treatment of an siRNA against this clone revealed that depletion of hCenexin results in a potent mitotic arrest with multiply segregating, but still connected, chromosome morphologies. In a separate study, we observed that the transfected Clone #17-encoded protein co-localizes with CREST antigen, an established kinetochore marker, at both the interphase and mitotic kinetochores. Consistent with the two-hybrid analyses, immunoprecipitation of endogenous Plk1 co-precipitated both of these proteins from transfected HeLa cell lysates. We also found that these proteins directly interact with endogenous Plk1 in vivo under physiological conditions. Whether these interactions are important for the localization and function of Plk1 at the respective centrosome/kinetochores is currently under investigation. We will examine whether these proteins serve as substrates or upstream regulators of Plk1 by carrying out both in vitro kinase assays and in vivo co-transfection experiments. For the centrosome-localizing protein, we will further investigate whether the observed pre-anaphase arrest is the result of an earlier defect in the cell cycle, such as defects in centrosome maturation or separation processes. For the kinetochore-localizing protein, studies will be carried out to determine whether it is required for spindle checkpoint function or kinetochore assembly/function.